The overall goal of this proposal is to integrate our mechanistic understanding of the signaling processes that take place in the outer segment of photoreceptor cells with the framework of the cellular processes responsible for maintenance of the protein composition of this organelle. To this end, Aim 1 will continue our longstanding investigation of the GTPase activating protein complex for transducin with an emphasis on the interplay between the cellular and catalytic aspects of its function. In Aims 2 and 3 a similar interplay will be analyzed for rhodopsin, which has three distinct roles in photoreceptor cells: signaling (activation of the phototransduction cascade), structural (building material of the disc membranes) and targeting (directing the flow of vesicles transporting rhodopsin and likely other proteins, e.g. the GTPase activating complex, to the outer segment). We will first test whether the role of rhodopsin in directing outer segment vesicular transport can be dissociated from its other major functions and then determine whether the structural function of rhodopsin can be replaced by other membrane proteins made to localize to the photoreceptor discs. Finally, Aim 4 will elucidate trafficking pathways responsible for the delivery of other proteins functioning in the outer segment. We will focus on a photoreceptor disc rim protein, peripherin-2/RDS, which is the most likely known candidate for using an intracellular targeting mechanism alternative to rhodopsin. The proposed experiments are relevant to understanding the most basic issues in photoreceptor cell biology and are key for understanding the causes of many types of photoreceptor degeneration associated with defects in protein signaling, targeting and trafficking.